Insecticidal fermentation broth from actinomycetes containing enhanced ratio of active to inactive dunaimycins

ABSTRACT

Methods and compositions are provided for the preparation of insecticidal compositions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to and is a divisional of U.S. patentapplication Ser. No. 12/939,991, filed Nov. 4, 2010, which in turnclaims priority to U.S. Ser. No. 61/258,716, filed Nov. 6, 2009; andU.S. Serial No. 61/259,549, filed Nov. 9, 2009, the contents of each areincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

NOT APPLICABLE.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE.

BACKGROUND OF THE INVENTION

Insects cause over 22 billion dollars of crop damage in the UnitedStates alone. Many of the widely used insecticides are older neurotoxiccompounds, and there is a substantial need for new, safer chemistries.Strain NRRL No. 30232 is a proprietary strain of Streptomyces galbusdescribed in U.S. Pat. No. 6,682,925 that produces a group of closelyrelated macrolides, called dunaimycins, exhibiting insecticidalactivity, especially against Lepidoptera (caterpillars). Dunaimycins are24-membered macrolides produced by actinomycetes that were firstreported in the early 1990s by research groups at Abbott Laboratories.The Abbott researchers elucidated the structures of various dunaimycinspecies, A1, C1, C2, D2, D2S, D3 and D4S; studied their antimicrobialand immunosuppressive activities; and described the taxonomy of theproducing organisms, two strains of Streptomyces diastatochromogenes.Karwowski, J. P., et al. Journal of Antibiotics December 1991, p.1312-1317; Hochlowski, J. E., Journal of Antibiotics, December 1991, pp.1318-1330; and Burres, N. S., et al., Journal of Antibiotics, December1991, pp. 1331-1341. A few later publications described insecticidal oracaricidal activity of specific dunaimycins.

While the literature describes purification of specific dunaimycins andelucidation of their structure and activity, it lacks disclosuresregarding interactions between the various species of dunaimycins andcomparisons of insecticidal activity of the various dunaimycins. Inaddition, although production of fermentation broth containingdunaimycins in the gram per liter range is necessary to create acommercially viable insecticidal product, dunaimycin-relatedpublications describe production of dunaimycins at milligram per literlevels. The background literature does not disclose methods forincreasing dunaimycin production nor does it appreciate thecorresponding challenges of scale-up.

In conducting experiments to increase dunaimycin production ofStreptomyces galbus NRRL No. 30232 through screening of libraries ofantibiotic-resistant mutants and optimizing fermentation conditions,Applicants increased dunaimycin production to previously unreportedlevels of at least 2.5 gram dunaimycin per liter fermentation broth(prior to concentration). Surprisingly, however, Applicants found thatan increase in dunaimycin production was not necessarily proportional toan increase in insecticidal activity. Applicants analyzed in greaterdetail the interactions between the various species of dunaimycins anddetermined that particular dunaimycin species are insecticidally active,while others are either inactive or antagonistic to insecticidalactivity.

BRIEF SUMMARY OF THE INVENTION

Therefore, the present invention relates to compositions comprisingcultures of actinomycetes having an optimized ratio of insecticidallyactive to inactive dunaimycins. In one embodiment, such cultures containgram per liter levels of dunaimycins.

This invention also relates to processes for producing fermentationbroth enriched in insecticidally active dunaimycins by cultivatingactinomycetes capable of producing both active and inactive dunaimycinsin optimized culture media until at least about one gram totaldunaimycins per one liter fermentation broth is produced. In oneembodiment the optimized culture media contains carbon and nitrogensources in a C:N ratio of at least 10:1, by weight. The weight ratios ofC:N can be calculated by known methods based on the masses of C and N inthe overall mix of the fermentation medium. Accordingly, multiplecomponents can add different amounts to each of the C and N portions,but the overall ratio in the mixture will have the indicated ratio.

This invention also encompasses methods for producingdunaimycin-containing fermentation products using the above process andstandard purification techniques to obtain a pure or semi-purefermentation product enriched in insecticidally active dunaimycins.

Another aspect of this invention is a method for screening actinomycetesfor insecticidal activity by measuring the amount of active and inactivedunaimycins produced by a library of dunaimycin over-producingactinomycetes. In one embodiment of this method, the screening step ispreceded by a step in which a library of mutantdunaimycin-over-producing actinomycetes is created from a parentdunaimycin-producing strain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a general structure for the dunaimycins describedherein, including those identified as A1, C1, C2, C2S, D2, D2S, D3, D3Sand D4S. See descriptions of activities of various dunacimycins on pages5-6 and in Table 1 of Example 2 on page 14.

FIG. 2 provides a numbering scheme for the dunaimycins described herein.

FIG. 3 provides the results for the bioactivity of a Rifampcin-resistantmutant vs. wild-type stage 1 screening assay.

FIG. 4 provides the results for the bioactivity of a Rifampcin-resistantmutant vs. wild-type stage 3 screening assay, with LD50 data.

FIG. 5 provides the results of a HPLC determination of AI ratios ofRifampicin-resistant mutant stage 3 screening.

FIG. 6 provides specific structures of dunaimycins referred to herein asA1, C1, C2, C2S, D2, D2S, D3S and D4S. The structure of the dunaimycinreferred to herein as D3 is not provided in FIG. 6 but can be determinedfrom FIG. 1.

FIG. 7 provides a graph showing macrolide production for a typical batchprocess for AQ6047 with wild-type.

FIG. 8 provides a graph showing macrolide production for a typicalfed-batch process for AQ6047 with wild-type.

FIG. 9 provides a graph showing macrolide production for a typicalfed-batch process for AQ6047 with mutant.

FIG. 10 provides a graph showing dunaimycins production andcorresponding bioactivities of M1064 in fed-batch fermentation.

FIG. 11 provides a graph showing improvement in dunaimycins productionthrough fermentation optimization.

FIG. 12 provides a graph showing bioactivities against beet army worm byfermentation broth of RF 1694 and RF 1696 along with Javelin (a Bacillusthuringensis product) Spintor (a spinosyn-based product) and untreatedcontrol (UTC).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to insecticidal actinomycete-producedfermentation broths and related fermentation solids that contain anoptimized ratio of insecticidally active to inactive dunaimycins. Theterm “fermentation broth” refers to the culture medium resulting afterfermentation of a microorganism and encompasses the microorganism andits component parts, unused raw substrates, and metabolites produced bythe microorganism during fermentation, among other things. The term“fermentation solid,” as used herein, refers to concentrated and/ordried fermentation broth. The term “crude extract,” as used herein,refers to organic extracts of fermentation broth, such as ethyl acetateextracts. The term “semi-purified,” as used herein, refers tometabolites isolated from fermentation broth that are about 50% to about90% pure. The term “purified,” as used herein, refers to metabolitesthat are isolated from fermentation broth that are about 91% to about100% pure.

The actinomycete in the fermentation broth produces both insecticidallyactive and inactive dunaimycins and may be a strain of Streptomyces.Several previously identified strains of Streptomyces produce multiplespecies of dunaimycins. Such strains include Streptomyces galbus NRRLNo. 30232, described in U.S. Pat. No. 6,682,925 and Streptomycesdiastatochromogenes strains AB1691Q-321 and AB1711J-452, described inKarwowski, J. P., et al, Journal of Antibiotics December 1991, p. 1312.In addition, strains of Streptomyces can be readily screened for abilityto produce active and inactive dunaimycins using techniques describedherein and known in the art.

Dunaimycin-producing actinomycetes may also be mutants ofdunaimycin-producing parent strains, such as isolated wild type strains.Mutants may be obtained by physical and chemical methods known in theart. For example, mutant strains may be obtained by treatment withchemicals such as N-methyl-N-nitro-N-nitrosoguanidine. Spontaneousmutants may be obtained without the intentional use of mutagens by, forexample, classical methods, such as growing the parent strain in thepresence of a certain antibiotic to which the parent is susceptible andtesting any resistant mutants for improved biological activity or, inthis application, overproduction of dunaimycins compared to the parent.Mutants may also be obtained by producing protoplast fusions of strainsthat produce dunaimycins, using the techniques described in Keiser, T.,et al. Practical Streptomyces Genetics, 2000, pp. 57-58. Mutants thatproduce more dunaimycins than the parent strain are referred to hereinas “dunaimycin-overproducing strains.” In some embodiments, suchdunaimycin-overproducing strains produce about 2 to about 20 times moredunaimycins than the parent strain.

In one embodiment, such a mutant is Streptomyces galbus M1064. Thismutant was obtained by screening for antibiotic-resistant,dunaimycin-overproducing mutants of Streptomyces galbus NRRL No. 30232,as described in detail in Example 1. M1064 was deposited on Nov. 5, 2009in the USDA's Agricultural Research Service Patent Culture Collectionlocated at 1815 N. University Street Peoria, Ill. 61604 U.S.A. (NRRL) inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure andthe Regulations thereunder (Budapest Treaty) and was assigned AccessionNo. NRRL 50334. This strain has been deposited under conditions thatassure that access to the cultures will be available during the pendencyof this application. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The general structure of dunaimycins and various species of dunaimycinsare shown in FIG. 1. The term “active dunaimycin” as used herein refersto those dunaimycin species that are insecticidally active. In oneembodiment, purified active dunaimycin species are insecticidally activeagainst at least some insects at a concentration of less than about 1000ppm; in another at a concentration of less than about 500 ppm; in yetanother at a concentration of less than about 200 ppm. Activedunaimycins include D3S, C2S, D2, D2S, D4S and C2. The term “inactivedunaimycin” as used herein refers to those dunaimycin species that areinsecticidally inactive. Purified inactive dunaimycin species do notshow insecticidal activity against some or all insects and are inactiveup to about 1000 ppm. Inactive dunaimycins include A1 and C1. In oneembodiment, insecticidal activity is determined by activity againstLepidopterans. In another embodiment, active and inactive dunaimycinsare distinguished by the ability of each to show insecticidal activityagainst beet army worm with an LD50 of 200 ppm or less.

In some embodiments, active dunaimycins have a vinylogous enol ether orhemiacetal or acetal moiety at position C18-C19, as do D2S, C2S, D2, D2Sand C2. Numbering of carbons in the dunaimycins is shown in FIG. 2. Insuch embodiments, inactive dunaimycins lack these moieties and insteadhave a ketone at position C 19, as do A1 and C1. Without intending to bebound by any specific theory, Applicants believe that the presence ofthe vinylogous enol ether or the hemiacetal or acetal moiety is neededfor biological activity, while presence or absence of the amino sugarmoiety at the R₂ position shown in FIG. 2 is not essential to activitybut increases the molecule's water solubility. One further considerationis that the active dunaimycins bind agonistically (that is, asactivators) to specific protein targets in the insect while inactivedunaimycins competitively inhibit binding of the active species, butthemselves exhibit only weak effects on these protein targets.

In one embodiment, the fermentation broth includes at least about 0.5 gtotal dunaimycin per liter. In another embodiment, it includes at leastabout 1 gram total dunaimycin per liter. In yet another embodiment, itincludes at least 3 grams total dunaimycin per liter of broth. In stillanother embodiment, it includes at least 7 grams total dunaimycin perliter of broth. In another embodiment, the fermentation broth includesbetween about 1 gram and about 7 grams total dunaimycin per liter ofbroth.

In one embodiment, the optimized ratio of active to inactive dunaimycinsis at least about 1:1. In another embodiment, the optimized ratio ofactive to inactive dunaimycins is at least about 2:1. The ratio can becalculated based on either a molar or weight ratio of the dunaimycins.For example, dunaimycins A1 and C1 do not have a sugar component, whiledunaimycins D2S and D3S have an attached sugar. The sugar-containingdunaimycins are about 10% by weight heavier than those without sugar anddo not appreciably impact the overall ratios of active to inactivedunaimycins.

Compositions of the present invention include the above-describedfermentation broth, which may be used as is, or dried and/orconcentrated. The fermentation broth or fermentation solids may beformulated with one or more carriers. If necessary for the end use, thefermentation broth may be treated to inactivate the microorganism byheat, chemical, or irradiation means before formulation. Carriers areinert formulation ingredients added to the fermentation broth to improverecovery, efficacy, or physical properties and/or to aid in packagingand administration. Such carriers may be added individually or incombination. In some embodiments, the carriers are anti-caking agents,anti-oxidation agents, bulking agents, and/or protectants. Examples ofuseful carriers include polysaccharides (starches, maltodextrins,methylcelluloses, proteins, such as whey protein, peptides, gums),sugars (lactose, trehalose, sucrose), lipids (lecithin, vegetable oils,mineral oils), salts (sodium chloride, calcium carbonate, sodiumcitrate), and silicates (clays, amorphous silica, fumed/precipitatedsilicas, silicate salts). In some embodiments, the carriers are addedafter concentrating fermentation broth and during and/or after drying.

In one embodiment, the compositions include fermentation solids that areformulated as wettable powders or water dispersible granules in whichthe fermentation solid is present at a concentration of about 1% toabout 90% by weight.

In another embodiment, the compositions may be formulated asemulsifiable concentrates in which concentrated fermentation broth isdissolved in an inert carrier which is either a water-miscible solventor mixture of a water-immiscible organic solvent and emulsifiers. Theconcentrated fermentation broth is present at a concentration of about10% to about 90%.

In another embodiment, the compositions may be formulated as an aqueoussuspension, in which the concentrated fermentation broth or thefermentation solid is dispersed in an aqueous vehicle at a concentrationin the range of from about 0.1% to about 50% by weight.

Certain methods are useful for preparing fermentation broths having anoptimized ratio of active to inactive dunaimycins. In one aspect of thisinvention, the above-described fermentation broth is produced byculturing a dunaimycin-producing actinomycete in a suitable culturemedium optimized to enhance production of active dunaimycins and tominimize production of inactive dunaimycins. Suitable culture media maybe obtained by adjusting the ratio of nitrogen source to carbon source,such that there is more nitrogen available than carbon in the culturemedia. In one embodiment, the ratio of carbon to nitrogen is betweenabout 1:1 to about 200:1. In another, the ratio is between about 10:1 toabout 30:1. In another, the ratio is about 20:1. As noted in the Summaryabove, the indicated ratios are for the relative weight amounts of C andN in the sources. In one embodiment, a batch process is carried out inwhich the ratio is set during the batching (before startingfermentation) and is not adjusted during fermentation. In anotherembodiment, consumption of C and N sources is monitored and additionsare made to the fermentation medium during the process.

Selection of specific carbon and nitrogen sources may also be used tooptimize the ratio of active to inactive dunaimycins. Suitable carbonsources include monosaccharide, disaccharides, and polysaccharides, suchas glucose, fructose, sucrose, molasses, maltodextrin, and starch, andplant-derived fatty acids and oils such as soy oil and cotton seed oil.Suitable nitrogen sources may be microbial, animal and plant derivedproteins and amino acids or substances containing proteins. In oneembodiment, the nitrogen source comes from soy-derived products, such assoy flour, soytone, soy hydrolysate, and soy grits. In anotherembodiment the nitrogen source includes cotton-seed derived products,such as PROFLO (Traders Protein, Lubbock, Tex.) and martone (MarcorDevelopment Corporation, Carlstadt, N.J.), corn steep powder, maltextract, and potato extract. In yet another embodiment, the nitrogensources are animal-derived, such as casein derived products (skim milk,casein peptone, casein hydrolysate, casamino acids) and peptone. In yetanother embodiment, the nitrogen sources are microbial-derived, such asyeast extracts or whole yeasts. Other essential elements necessary forgrowth and development of the organism should also be included in theculture medium and will be well known to those of skill in the art.

Monitoring the levels of C and N in the fermentation medium can becarried out using conventional methods to monitor the carbon and/ornitrogen sources. For example, glucose levels can be monitored using aBeckman Glucose Analyzer, sucrose can be monitored using an enzymatic(invertase) method, while more complex sources (for example, molassesand malt extracts) can be analyzed by a colorimetric reducing sugarmethod after acid hydrolysis of a fermentation media aliquot.

Conventional large-scale microbial culture processes including submergedfermentation, solid state fermentation, or liquid surface culture may beused to cultivate dunaimycin-producing actinomycetes. Dunaimycins areproduced when grown at temperatures between about 20° C. and about 32°C. In one embodiment, pH of the fermentation broth is maintained betweenabout 6 and about 8.

In one embodiment, the dunaimycin-producing actinomycete is cultivateduntil at least about 1 gram dunaimycins per liter fermentation broth areproduced or, in another embodiment, until at least about 2 gramsdunaimycins per liter are produced, or in still another embodiment untilat least about 3 grams dunaimycins per liter fermentation broth areproduced, or in yet another embodiment until about 1 gram to about 7grams dunaimycins per liter are produced. Production of dunaimycins canbe following during the fermentation by testing extracts of the broth.One method for following production is analysis of extracts by highperformance liquid chromatography (HPLC).

In one embodiment, the above method for producing an insecticidalfermentation broth having an optimized ratio of active to inactivedunaimycins includes a further step of concentrating the broth byconventional industrial methods, such as centrifugation, membranefiltration, tangential-flow filtration, depth filtration, andevaporation. In some embodiments, the concentrated fermentation broth iswashed, for example via a membrane dialysis or ultra-filtration process,to remove residual fermentation broth.

In another embodiment, the fermentation broth or broth concentrate canbe dried with or without the addition of carriers using conventionaldrying processes or methods such as spray drying, freeze drying, traydrying, fluidized-bed drying, or drum drying. The resulting fermentationsolids may be further processed, such as by milling or granulation, toachieve a specific particle size or physical format.

In another aspect of the invention, the insecticidal fermentation brothproduced by the above process is chemically treated in order to modifythe inactive dunaimycins, so that they are unable to interfere orcompete with active dunaimycins for biologically relevant binding sitesor targets or so that they are transformed into active dunaimycins. Inone embodiment, selected dunaimycins can be removed from the mixture.For example, removal of dunaimycins A1 and C1 can be accomplished bypassing the entire mixture of all dunaimycins thru a resin with apendant amino functional group such as a primary amine, oxime,semicarbazide, or hydrazine. The C19-keto group present in A1 and C1will react preferentially with the pendant amino group and the remainingdunaimycins can be removed from the column by simply washing with anappropriate solvent. Dunaimycins containing, for example, the ossaminesugar moiety can be removed by passing the dunaimycin mixture thru acation exchange resin. The amino group present on the ossamine sugarwill help retain the active dunaimycin molecules having the sugar on thecolumn, and allow for the inactive dunaimycins to pass thru. The activeossamine sugar dunaimycins can then be eluted out of the resin withbasic buffer.

In another embodiment, inactive dunaimycins can be reduced in themixture by genetic manipulation of the actinomyces strains tospecifically knock out the expression of the inactive dunaimycin eitherby classical genetics or by molecular means.

In yet another aspect of the invention, the insecticidal fermentationbroth produced by the above process is subjected to standardpurification techniques in order to separate active dunaimycins frominactive dunaimycins and obtain semi-purified or purified dunaimycins.

The present invention also encompasses methods for screeningactinomycetes for the ability to preferentially produce activedunaimycins. In one embodiment the ratio of active dunaimycins toinactive dunaimycins produced by actinomycetes of interest is greaterthan about 1:1. In another embodiment, no inactive dunaimycins areproduced. In another aspect of the invention, the screening step ispreceded by generation of a library of dunaimycin-overproducing strains.For example, Examples 1 and 2 describe producing a library of mutantsand using chromatography methods to identify those that over-producedunaimycins. Other methods for rapid screening of mutants that areover-producers of dunaimycin include creating a colorimetric reporterstrain.

The compositions of this invention are useful for control of insects.Therefore, a further aspect of the present invention is directed tomethods for controlling an insect by applying to the locus of the insectan effective amount of the compositions of the present invention. Asused herein, the term “control” or “controlling” means to kill insectsor to decrease the number of viable insect eggs. The term “locus” meansthe environment in which the insect lives or where its eggs are present,such as plant parts or the area surrounding plants which the insectmight eat or inhabit. An “effective amount” is the amount needed tocause a measurable reduction of the treated insect. In some embodiments,greater than about 50% control is obtained, in others greater than about60%; in others greater than about 70%; in others greater than about 75%;in others, greater than about 80%.

In one embodiment, rates of about 100 ppm to about 10000 ppm offermentation solids per acre are used. In another embodiment, betweenabout 1 and about 50 g of formulated fermentation solids are applied peracre.

In one embodiment, the compositions are used to control Lepidoptera,such as tobacco budworm (Heliothis virescens), beet armyworm (Spodopteraexigua), cabbage looper (Trichoplusia ni) and diamondback moth (Plutellaxylostella). Other typical Lepidoptera are Egyptian cotton leaf worm,oblique banded leafroller, black cutworm, pandemis leafroller, codlingmoth, fall armyworm, and corn earworm.

EXAMPLES Example 1 Production of Mutant

QST6047 (Steptomyces galbus NRRL No. 30232) is a wild strain ofStreptomyces galbus that produces a suite of dunaimycins. Thedunaimycins produced by QST6047 have insecticidal activity againstLepidoptera. With the goal to increase dunaimycin production andbioactivity, a dunaimycin over-producing mutant M1064 was created fromthe wild strain QST6047 through an antibiotic-resistant mutant screeningprogram in which libraries of mutants resistant to individualantibiotics (gentamicin, rifampicin, streptomycin, paromomycin ortobramycin) were produced. A detailed description of creation andscreening of the rifampicin-resistant library, from which adunaimycin-overproducing strain was ultimately selected for furtherdevelopment, is described below.

Spores from a SFM (Mannitol 20 g/L, soy flour 20 g/L, agar 20 g/L) plateculture of S. galbus QST6047 suspended in 20% glycerol were heat shockedfor ten minutes in a 50° C. water batch. 100 μl of the spore suspensionwas then plated onto GYM (glucose 4 g/L, yeast extract 4 g/L, maltextract 10 g/L, and agar 12 g/L) supplemented with 5 μl/ml rifampicin.Enough spore suspension was plated in order to have at least 300individual colonies to isolate, purify, and screen.

Agar plugs containing rifampicin-resistant bacteria were used toinoculate culture tubes that contained 31-3C medium (Proflo 20 g/L, maltextract 20 g/L, KH₂PO₄ monobasic 6 g/L, K₂HPO₄ dibasic 4.8 g/L) andgrown for six days at 28 C. The culture broth was then serially dilutedand tested for bioactivity using a beet army worm egging bioassay (BAWEgging Bioassay) as follows. The test samples were distributed across a96-well microplate containing beet army worm (BAW) eggs. The each wellof the microplates contained diet and about known number of beet armyworm eggs. The microplate was incubated under optimal conditions for theeggs to hatch. After seven days, the number of worms still living werecounted and a break point rating was assigned. The break point of themutant relative to the break point of the wild type was noted. Eachbreak point represented a 2× increase in activity over wild type. Anymutant exhibiting bioactivity above the wild type was then cultured inshake flasks. Of 300 mutants screened, 20 showed increased bioactivity(FIG. 3).

Selected mutants with higher bioactivity and ability to sporulate onagar plates were grown in 1-L baffled shake flasks and subsequentlyscaled up to 20-L bioreactors containing 31-3C media. Both bioactivity(FIG. 4) and dunaimycin production (FIG. 5) were measured for eachmutant. Surprisingly, although the mutants produced higher levels ofdunaimycins than the wild type, the bioactivity did not increaseproportionally.

Example 2 Identification of the Potency Differences in Bioactivities ofVarious Dunaimycins (HPLC Identification and Bioassays)

The various species of dunaimycins were isolated from either a mutantcalled M1064 or wild type (Streptomyces galbus QST6047) for analysis ofactivity. Either M1064 or wild type was grown up in 2 L flasks using themedia described above. The whole broth culture was extracted twice with1 L ethyl acetate and the combined organic extract concentrated underreduced pressure. The crude extract was loaded onto a silica gel columnequilibrated in hexanes, and the column was eluted with hexanes followedby a step solvent gradient of hexanes-ethyl acetate. The fractionscontaining the dunaimycins were determined by analytical HPLCreverse-phase C8 column and BAW egging bioassay.

Active fractions were further fractionated using a preparativereverse-phase C8 column [Zorbax Exclipse XDB-C8, 5 μm; 9.4×25 cm at aflow rate of 2.0 mL/min and UV detection at 220 nm. Eluting solventsystem consisted of water (10 mM NH₄OAc):methanol mixture] which yieldedpure dunaimycins. All isolated dunaimycins were confirmed using UV andmass spectrometry. Structures of identified dunaimycins are shown inFIG. 6.

The separated, identified dunaimycins were then tested for bioactivityagainst beet armyworm eggs as follows. Stock solutions were provided as15% aqueous ethanol solution of various semipurified dunaimycins, eitheralone or in combinations. For reference purposes, Javelin WDG (Bacillusthuringiensis subspecies Kurstaki; ThermoTrilogy Inc.) was weighed outand diluted to give a 1,000 ppm stock solution (positive chemicalstandard). These stock solutions were transferred to a deep wellmicrotitre plate: 1.4 ml of each stock solutions were placed in the 2 mlwell across the top row (A wells 1-12). A Packard Multiprobe II liquidhandling system (Perkin Elmer Inc.) then carried out a dilution program,first adding 700 μls of DI H₂O to the remaining 84 wells (B-H 1-12). Thefour tip arm then performed a sequence of aspiration and dispensingsteps to mix the samples and then transferred them into an adjoiningwell to give eight 50% serially diluted samples containing 700 μl perwell. The robot then used a transfer program to pipette all of theserial diluted samples to labeled 96 well plates. Aliquots containing 40μl of each dilution were dispensed into six wells across a microtitreplate containing ˜200 μl of artificial diet/well. Two samples weretested on every 96 well plate; Sample one at the stock concentration inwells A1-6; sample two into wells A7-12. Row B contains a 50% dilutionseries or a ½ × solution of sample one in row B wells 1-6 and sample twoin row B wells 7-12. The last two samples in each set were the chemicalstandard and untreated controls containing DI water. The samples weredried rapidly at room temperature under forced air and then each wellwas egged with 5-10 synchronized beet army worm eggs in agar using arepeating pipette. The egg:agar mixture is dried under forced air andthe plates were covered with perforated Mylar. The plates were incubatedat 28° C. on a 16:8 light: dark cycle. After five to seven days thenumber of live insects in each row was tabulated. Mortality was recordedas the number dead over the number treated and expressed as controlcorrected percent mortality. The data was exported from Excel into PoloPC (LeOra Software 1987) and LC 10, 50, and 90s were calculated usingnatural response and 95% confidence intervals. Results are shown inTable 1.

TABLE 1 Bioactivity of purified dunaimycins and combinations LD50 (ppm)BAW Compound Ratio assay Dunaimycin A1 Tested alone Inactive up to 1000ppm Dunaimycin C1 Tested alone Inactive up to 1000 ppm Dunaimycin C2Tested alone 31 Dunaimycin C2S Tested alone 10 Dunaimycin D2/D2S Testedalone 11 Dunaimycin D3S Tested alone 9 A1:D2/D2S 1:1 107.8 A1:D2/D2S 4:1150.1 A1:D2/D2S 8:1 239.6 D2/D2S:C2:C1:A1 2.6:1:3.75:5.1 156.1 A1:C1 1:1Inactive up to 1000 ppm D2/D2S:C2 2.57:1   25.1

Example 3 Fermentation Optimization (Including Fermentation and RelatedBioassays)

Initial fermentation optimization studies focused on improving thefermentation process for QST6047 and for the mutant M1064 to increasetotal dunaimycin production, as described below. (Further studies withM1064, also described below, focused on optimizing production of activedunaimycins.) Improvements associated with strain improvement andfermentation optimization studies were tracked simultaneously bybioassays for insecticidal activity and by HPLC for dunaimycinproduction. The bioassay employed a MultiPROBE® II automated liquidhandling system, and a 96-well-plate assay using beet armyworm on anartificial diet followed by Probit analysis of the raw data. Thebioactivity is reported as the lethal concentration of sample needed tokill 50% of the worm population (LC50). The HPLC assay utilized apartition extraction of the whole broth followed by C-18 reverse phasechromatography and peak integration analysis (mAU).

Under optimal temperature, pH, aeration and agitation, a three foldincrease in the QST6047 strain's dunaimycin production was achieved overthe original batch fermentation process. A profile of typical growthparameters and dunaimycin production for QST6047 in an optimized batchprocess is presented in FIG. 7. (In the Figures the term macrolide isequivalent to dunaimycin and the term lead candidate is equivalent toM1064.) To further improve macrolide production, a fed-batch process wasdeveloped. Several different feeding strategies were investigated. Aprocess was developed in which carbohydrate was batched duringsterilization and a variable feed rate with additional carbohydrate andnitrogen was initiated later on in the process to maintain residualcarbohydrate levels at concentrations less than 1 g/L. This fed-batchprocess resulted in a two fold increase in the QST6047 strain'sdunaimycin production over the optimized batch process. A profile oftypical growth parameters and macrolide production for the wild-typeoptimized fed-batch process is presented in FIG. 8.

Fermentation conditions were also optimized for increased dunaimycinproduction of M1064. Media rebalancing and refining of batch processparameters was necessary to determine the optimum conditions formacrolide production, and a 1.5 fold increase in macrolide productionover the wild-type batch process was achieved. The most significantincrease in macrolide production was achieved with M1064 by employing afed-batch process. Similar to the wild-type fed-batch process, somecarbohydrate was initially present at the start of the process. Variablefeed rates with additional carbohydrate and nitrogen was initiated lateron in the process to maintain residual carbohydrate levels atconcentrations less than 1 g/L. Slower feed rates than with thewild-type were used due to a slower carbohydrate consumption rate by themutant. With a fed-batch process, a 13 fold increase in dunaimycinproduction was achieved over the original wild-type batch process. Aprofile of typical dunaimycin production, growth parameters, andcarbohydrate utilization for a fed-batch process with M1064 is presentedin FIG. 9. A profile of dunaimycin production and the correspondingbioactivity for M1064 is presented in FIG. 10. FIG. 11 shows theincrease in dunaimycin production during the optimization process.

Additional media development and process studies were conducted toincrease bioactivity of M1064 by optimizing the ratios of activedunaimycins and inactive dunaimycins. Various carbon and nitrogensources were used to culture the mutant M1064 and the wild type. It wasfound that different carbon and nitrogen sources can have differenteffects on the production of active and inactive dunaimycins and thatrelative ratio between active and inactive can have significant impacton the insecticidal activity. Glucose and soy flour were the carbon andnitrogen sources particularly useful for improving the ratios betweenactive to inactive dunaimycins. See Tables 2 and 3 below.

TABLE 2 % Active % Inactive Ratio Carbon Dunaimycins DunaimycinsActive/Inactive LC50 Sucrose 31 41 0.76 0.26 Glucose 43 20 2.15 0.15Molasses 31 43 0.72 0.90 Malt 33 51 0.64 0.65 Extract

TABLE 3 % % Ratio Active Inactive Active/ Condition Culture DunaimycinsDunaimycins Inactive LC50 Batch WT 41 28 1.46 0.31 w/o soy flour Batchw/ WT 48 24 2.0 0.26 soy flour Batch M1064 33 50 0.66 0.65 w/o soy flourBatch w/ M1064 36 11 3.27 0.24 soy flour

After identifying optimal sources for carbon and nitrogen, furtherbioreactor studies were focused on the optimization of the fermentationprocess, particularly on the batch and fed-batch processes. It wasdiscovered that, in addition to the sources of carbon and nitrogen, theC:N ratio of the fermentation media also had significant impact on thelevel of active dunaimycin production and the bioactivity.

TABLE 4 C:N ratios of the batch and fed-batch fermentation processesFermentation process Nitrogen (g) Carbon (g) C:N ratio Batch 2.7 53.120:1 Fed-batch 1.9 24.3 13:1

The following are descriptions of the optimized batch and fed-batchprocesses for M1064 using the above C:N ratios.

Batch Fermentation Process Seed Train

The first stage seed was prepared by thawing a frozen vial of theculture and dispensing half of its contents into two, 250 ml baffledflasks containing 50 ml of sterile 31-3C seed media (Table 5). Theculture was incubated at 28° C. on a shaker at 220 rpm for three days.

The second stage seed was prepared by transferring 10 ml of the threeday old first stage seed to a 2-liter baffled flask containing 250 ml of31-3C seed media. The cultures were incubated at 28° C. on a shaker at220 rpm for three days. Three 2-liter flasks were prepared for eachfermentation which had a working volume of 15 liters.

Fermentation Media and Conditions

The media ingredients for the 15 L fermentation process were weighed andsuspended in the bioreactor in 14-liters of distilled water. The mediaingredients for the M1064 process 352 are presented in Table 6. Themedia was thoroughly mixed and the pH adjusted to 7.3. Aftersterilization, the media was re-adjusted to pH 6.8.

Fermentation Process—20-Liter Bioreactor

The contents from three 2-liter flasks (750 ml) of three-day-oldsecond-stage-seed was pooled and used to inoculate a sterile bioreactor.The cultures were streaked onto nutrient agar to confirm culture purity.The pH was controlled at 6.8 throughout the fermentation. Thecultivation temperature was 25° C. and the rate of aeration was 15L/min. The dissolved oxygen concentration was controlled at 50% with anagitation cascade between 300 and 850 rpm. The fermentation wasconducted for 90-95 hours with samples taken twice a day to monitor theprocess.

Analytical Methods for Growth, Dunaimycin and Bioactivity

Growth was monitored by % solids, % cell dry weight and occasionallycolony forming units. Glucose concentration was monitored using theBeckman glucose analyzer.

The total dunaimycins present per ml of whole broth was quantified byHPLC. Sample extracts were prepared by combining 5 ml of whole brothwith 3 ml of ethyl acetate and 2 g of magnesium sulfate.

Bioactivity of the whole broth was measured by using the BAW eggingbioassay described in Example 1. Javelin was run as the positive controlat an initial concentration of 1000 ppm. The LD50 was then calculated.

TABLE 5 Seed Media (31-3C) Ingredient g/L Proflo cottonseed meal 20.0Malt Extract 20.0 KH₂PO₄ 6.0 K₂HPO₄ 4.8

TABLE 6 Fermentation Media for Process M1064 Ingredient g/L Proflocottonseed meal 20.0 Glucose 50.0 Soyflour 10 Soy Oil 5.0 Sigma 204Antifoam 1.0

Fed-Batch Fermentation Process Seed Train

The first stage seed was prepared by thawing a frozen vial of theculture and dispensing half of its contents into two, 250 ml baffledflasks containing 50 ml of sterile 31-3C seed media (Table 7). Theculture was incubated at 28° C. on a shaker at 220 rpm for three days.

The second stage seed was prepared by transferring 10 ml of the threeday old first stage seed to a 2-liter baffled flask containing 250 ml of31-3C seed media. Three 2-liter flasks were inoculated for eachfermentation run which had a working volume of 15 liters. The cultureswere incubated at 28° C. on a shaker at 220 rpm for three days.

Fermentation Media and Conditions

The media ingredients for the 15 L fermentation process were weighed andsuspended in the bioreactor in 14-liters of distilled water. The mediaingredients for the 6047 wild type process 328 are presented in Table 8.The media was thoroughly mixed and the pH adjusted to 7.3. Aftersterilization, the media pH was re-adjusted to 6.8.

Fermentation Process—20-Liter Applikon Bioreactor

The contents from three 2-liter flasks (750 ml) of three-day-oldsecond-stage-seed were pooled and used to inoculate the sterilebioreactor. The cultures were streaked onto nutrient agar to confirmculture purity. The pH was controlled at 6.8 throughout thefermentation. The cultivation temperature was 25° C. and the rate ofaeration was 15 L/min. The dissolved oxygen concentration was controlledat 50% with an agitation cascade between 300 and 850 rpm. Thefermentation was conducted for 90-95 hours with samples taken twice aday to monitor the process using the analytical methods described below.

Feeding Regime

A feed of 30% glucose was initiated at the start of cultivation. Thegoal was to aggressively feed glucose such that little to no glucose wasdetected in the bioreactor. After 24 hours, a feed of 30% glucose and 3%monosodium glutamate was implemented. Feed rates were based on glucoseconsumption and increased or decreased based on the residual glucoseconcentration of the media.

Analytical Methods for Growth, Dunaimycin Production and Bioactivity

Growth was monitored by % solids, % cell dry weight. Cell dry weight ofthe washed pellet was monitored by using a Saterius moisture balance.Glucose concentration was monitored using the Beckman glucose analyzer.

The total dunaimycins present per ml of whole broth was quantified byHPLC. Sample extracts were prepared by combining 5 ml of whole brothwith 3 ml of ethyl acetate and 2 g of magnesium sulfate.

Bioactivity of the whole broth was measured against beet army worm usingthe BAW egging bioassay described in Example 1 in the Batch fermentationprocess section of this example.

TABLE 7 Seed Media (31-3C) Ingredient g/L Proflo cottonseed meal 20.0Malt Extract 20.0 KH₂PO₄ 6.0 K₂HPO₄ 4.8

TABLE 8 Ingredient g/L Fermentation Media for M1064 Proflo cottonseedmeal 20.0 Malt Extract 20.0 Soy Oil 5.0 Sigma 204 Antifoam 1.0 GlucoseFeed Stock Dextrose 300.0 Monosodium Glutamate Feed Stock Ingredientg/200 ml Monosodium 60 glutamate

Bioassay results obtained following fermentation optimization (includingstudies regarding C:N ratio) confirmed that bioactivity of M1064 wassubstantially increased compared to the wildtype 6047. The strainimprovement and optimized fed batch process described above gave rise toa 13 fold increase in dunaimycin production compared to the originalfermentation process used with 6047 and to a 35 fold increase inbioactivity, based on BAW egging assay, as compared to whole broth fromthe original fermentation process with 6047.

Example 4 Bioactivity Comparison of M1064 Grown with and without SoyFlour

Batch fermentation RF 1694 with the addition of soy flour and RF 1696without the soy flour were conducted on M1064 under the same conditionsdescribed at the end of Example 3. Whole broth from both fermentationruns were analyzed for dunaimycin production (Table 9) by HPLC and forinsecticidal activities on beet army worm using a leaf disc assay (FIG.12). The leaf disc assay was performed on lima bean leaves. The wholebroth from RF1694 with soy flour had higher bioactivity than RF1694 atboth the 1:32 and the 1:64 dilutions in the bioassay. The assay wasperformed with untreated control (UTC) and along with commercialinsecticide Javelin (a Bt based product) and Spintor (Dow AgroSciences;a spinosyn-based product).

Although the total dunaimycin produced was similar between the two runs,the amounts of active and inactive dunaimycins were different. Less ofthe inactive forms of dunaimycin was produced in the batch RF 1694 whichcontained soy flour, which resulted in higher ratio of active toinactive.

TABLE 9 Active and inactive dunaimycins produced in bioreactors. RatioTotal Active Inactive Active:Inactive RF1694 With 648009 224672 1698321.32 Soy flour RF1696 Without 549549 210613 210857 0.99 Soy flour

What is claimed is:
 1. A process for producing a fermentation product enriched in insecticidally active dunaimycins comprising cultivating an actinomycete that produces active dunaimycins and inactive dunaimycins in a culture medium containing carbon sources and nitrogen sources until at least about 0.5 milligrams of active dunaimycins per liter culture medium is produced, wherein the ratio of carbon to nitrogen sources is at least about 10:1 by weight.
 2. The process of claim 1, wherein the carbon sources are selected from the group consisting of monosaccharides, disaccharides, polysaccharides and complex carbohydrates from plant or animal materials.
 3. The process of claim 1, wherein the carbon sources are selected from the group consisting of sucrose, glucose, maltose, starch, maltodextrin, molasses and malt extract.
 4. The process of claim 1, wherein the nitrogen sources are selected from the group consisting of proteins and amino acids from plants or animals, soy flour, soytone, peptone, soy hydrolysate, soy grits, cotton seed flour, corn steep powder and potato extract.
 5. The process of claim 1, wherein the carbon source is glucose and the nitrogen source is soy flour.
 6. The process of claim 1, wherein the carbon source is glucose and the nitrogen source is a combination of cotton seed flour and soy flour.
 7. The process of claim 1, further comprising an initial step of screening a library of dunaimycin over-producing actinomycetes for production of active dunaimycins and inactive dunaimycins.
 8. The process of claim 1, further comprising purifying active dunaimycins from the fermentation broth and biomass.
 9. The process of claim 1, further comprising concentrating the fermentation broth and biomass.
 10. The process of claim 1, further comprising drying the fermentation broth and biomass.
 11. The process of claim 1, wherein the ratio of active dunaimycins to inactive dunaimycins is at least about 1:1.
 12. A fermentation product produced by the process of any of claims 1 to
 11. 